The expression of CD154 (CD40 ligand), a member of the Tumor Necrosis Factor (TNF) gene family, by activated T lymphocytes is critical in the development of humoral and cell-mediated immunity (Foy, et al. (1996) Annu. Rev. Immunol. 14:591-617; Grewal & Flavell (1998) Ann. Rev. Immunol. 16:111-135; Hollenbaugh, et al. (1994) Immunol. Rev. 138:23-37; Noelle (1996) Immunity 4:415-419). CD154 blockade retards the development and progression of immune responses in an array of transplantation and autoimmune disease models ranging from Systemic Lupus Erythematosus to Rheumatoid Arthritis to Multiple Sclerosis (Foy, et al. (1996) supra; Grewal & Flavell (1998) supra). Resting T cells express little or no CD154 (Lane, et al. (1992) Eur. J. Immunol. 22:2573-2578; Nusslein, et al. (1996) Eur. J. Immunol. 26:846-850; Roy, et al. (1993) J. Immunol. 151:2497-2510) and signals (anti-CD3, mitogenic lectins) that trigger resting T cells to engage in high levels of proliferation and cytokine production, elicit little (CD4+ T cells) or no (CD8+ T cells) expression on either mouse or human T cells (Lane, et al. (1992) supra; Nusslein, et al. (1996) supra; Roy, et al. (1993) supra), suggesting different pathways of gene regulation. Maximal expression of CD154 requires pharmacologic stimulation provided by phorbol myristate acetate (PMA) and calcium ionophores such as ionomycin (Lane, et al. (1992) supra; Nusslein, et al. (1996) supra; Roy, et al. (1993) supra; Roy, et al. (1994) Eur. J. Immunol. 25:596-603). Cyclosporine and glucocorticoids block CD154 induction on T lymphocytes; these effects are presumed to be transcriptional (Fuleihan, et al. (1994) J. Clin. Invest. 93:1315-1320; Roy, et al. (1993) supra), based on the presence of NF-AT sites in the CD154 promoter (Schubert, et al. (1995) J. Biol. Chem. 15:29264-29627). Since cyclosporine and glucocorticoids also inhibit cytokine production (Ashwell, et al. (1992) Ann. Rev. Immunol. 18:309-345; Sigal & Dumont (1992) Ann. Rev. Immunol. 10:519-60), this pathway does not account for the differential regulation of CD154 expression by T lymphocytes.
CD154 mRNA has been shown to be unstable in activated T lymphocytes, with a half-life (−30 minutes) approximating that seen with interleukins-2 (IL-2; Ford, et al. (1999) J. Immunol. 162:4037-4044; Murakami, et al. (1999) J. Immunol. 163:2667-2673; Rigby, et al. (1999) J. Immunol. 163:4199-4206; Suarez, et al. (1997) Eur. J. Immunol. 27:2822-2829). Nevertheless, several studies indicate that cytokine (IL-2 and TNF-alpha) and CD154 mRNA stability and expression are regulated through distinct pathways (Ford, et al. (1999) supra; Lindsten, et al. (1989) Science 244:339-343; Murakami, et al. (1999) supra). A region (nucleotides 468-835 referenced to the translational stop site) within the 986 nucleotide human CD154 3′-untranslated region (3′-UTR) confers an increase in the rate of mRNA turnover to chimeric reporter gene constructs in vivo (Hamilton, et al. (2003) Mol. Cell. Biol.; 23(2):510-25). This region lacks canonical AURE-type sequences, containing a polypyrimidine rich element as well as CA-dinucleotide repeat and polycytidine sequences. Members of the human polypyrimidine tract binding protein (PTB) gene family were identified and shown to directly interact with cytidines and uridines within this region (Hamilton, et al. (2003) supra; kosinski, et al. (2003) J. Immunol. 170(2):979-88), consistent with the presence of multiple PTB consensus binding sites (Anwar, et al. (2000) J. Biol. Chem. 275:34231-34235; Singh, et al. (1995) Science 268:1173-1176). Overexpression of splice isoforms of the PTB proteins differentially regulates CD154 expression and mRNA accumulation in a 3′-UTR-dependent manner in cell lines and normal human T cells. These effects are specific and restricted to reporter constructs containing the 3′-UTR polypyrimidine rich region.
The murine CD154 (mCD154) 3′-UTR is ˜0.3 kb shorter than its human counterpart, due to a 292 nucleotide insertion present at the 5′ end of the human 3′-UTR. The remaining portion of the human and entire mCD154 3′-UTR exhibits 70% conservation with retention of the polycytidine, polypyrimidine, and CA-dinucleotide repeat regions as well as an AURE that is found immediately 5′ of the polyadenylation signal sequence. Murine CD154 3′-UTR inhibits luciferase mRNA accumulation and protein activity in a comparable manner relative to that seen with the human CD154 3′-UTR. Further, deletion of the polypyrimidine-rich region cis-acting element enhances inhibition of 3′-UTR-dependent gene expression.
A novel pathway has now been identified which regulates translation of CD154 mRNA.